Systems and methods for analyzing body fluids

ABSTRACT

Systems and methods analyzing body fluids contain cells including blood, bone marrow, urine, vaginal tissue, epithelial tissue, tumors, semen, and spittle are disclosed. The systems and methods utilize an improved technique for applying a monolayer of cells to a slide and generating a substantially uniform distribution of cells on the slide. Additionally aspects of the invention also relate to systems and method for utilizing multi-color microscopy for improving the quality of images captured by a light receiving device.

PRIORITY CLAIM

This application is a divisional application of U.S. Ser. No.12/768,633, filed Apr. 27, 2010, which application claims the benefit ofpriority to U.S. Provisional Application 61/173,186, filed Apr. 27,2009, and is also a continuation-in-part of U.S. Ser No. 12/430,885,filed Apr. 27, 2009, which claims the benefit of priority to U.S.Provisional Application 61/047,920, filed Apr. 25, 2008, each of whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to a system and process for determiningcomposition and components of fluids. More specifically the presentinvention provides improved techniques for viewing cellular morphology,and determining the number of a particular type of cell in a portion ofa body fluid.

BACKGROUND OF THE INVENTION

Pathology is a field of medicine where medical professionals determinethe presence, or absence of disease by methods that include themorphologic examination of individual cells that have been collected,fixed or air-dried, and then visualized by a stain that highlightsfeatures of both the nucleus and the cytoplasm. The collection of thecells often involves capturing a portion of a person's body fluid,placing the body fluid on a slide, and viewing the fluid on the slideusing a microscope.

One of the most commonly performed pathologic studies is the CBC (theComplete Blood Count). To perform a CBC, a sample of blood is extractedfrom a patient and then the cells are counted by automated or manualmethods. The CBC is commonly performed by using an instrument, based onthe principal of flow cytometry, which customarily aspiratesanticoagulated whole blood and divides it into several analysis streams.Using the flow cytometer a number of primary and derived measurementscan be determined including: i) red blood cell (RBC) count, hemoglobin(Hb), hematocrit (Hct), red blood cell indices (mean corpuscular volume,MCV, mean corpuscular hemoglobin, MCH and mean corpuscular hemoglobinconcentration MCHC), red blood cell distribution width, enumeration ofother red blood cells including reticulocytes and nucleated red bloodcells, and red blood cell morphology; ii) white blood cell (WBC) countand WBC “differential” count (enumeration of the different normal whiteblood cell types, including neutrophils, lymphocytes, eosinophils,basophils and monocytes, and the probable presence of other normal andabnormal types of WBC that are present in various disease conditions);iii) platelet count, platelet distribution widths and other features ofplatelets including morphological features; and iv) other abnormal cellsor other unusual cells or cellular components that may be in circulatingblood. In flow cytometers, red blood cell, WBC, and plateletmorphological characterizations are typically made indirectly, based onlight absorption and light scattering techniques and/or cytochemicallybased measurements. Some advanced flow cytometers calculate secondaryand tertiary measurements from the primary measurements.

Flow based CBC instruments generally require extensive calibration andcontrol, maintenance, and skilled operators, and they have substantialcosts associated with acquisition, service, reagents, consumables anddisposables. One significant problem with these systems in routine useis that a large proportion of blood specimens require further testing tocomplete the assessment of the morphologic components of the CBC. Thisinvolves placing a sample of blood on a slide, smearing the sampleagainst the slide to form a wedge smear, and placing the slide under amicroscope. This process is often done manually by skilled medicaltechnologists, which increases the cost and time to receive results fromthe tests. The direct visualization of blood cells on a glass slide mustbe performed whenever the results of the automated test require furtherexamination of the blood sample. For example, a “manual” differentialcount is performed by direct visualization of the cells by anexperienced observer whenever nucleated immature RBCS are found or WBCssuspicious for infection, leukemias or other hematologic diseases arefound.

The proportion of these specimens requiring further review generallyranges from 10% to 50%, depending on the laboratory policy, patientpopulation and “flagging” criteria, with a median rate of around 27%.The most frequent reasons for retesting include the presence ofincreased or decreased number of WBCs, RBCs or platelets, abnormal celltypes or cell morphology, clinical or other suspicion of viral orbacterial infections.

In addition to additional work involved in performing manualdifferential counts, this process has a number of additional technicallimitations. These include distortions of cell morphology because ofmechanical forces involved in smearing the cells onto the slide, andcells overlapping one another, which makes visualization of individualcell morphology difficult.

Cytopathology is a subspecialty of pathology where medical professionalsdetermine the presence, or absence of disease by the morphologicexamination of individual cells that have been collected, fixed orair-dried, and then stained by a unique stain that highlights featuresof both the nucleus and the cytoplasm.

Examples of cytologic examination include the assessment of cellscollected from the uterine cervix (The Pap Test), evaluation of urinesamples for bladder cancer, assessment of lung samples for the presenceof cancer or inflammatory diseases, assessment of aspirates frompotential tumor sites, or the evaluation of samples collected fromeffusions in body cavities.

Cells that are collected for cytologic examination may be directlysmeared onto a glass microscope slide, they may be deposited onto theslide by centrifugation, they may be collected by a filtration method,or they may be concentrated by liquid-based cytology methods such as theThinPrep or SurePath methods. These approaches used different types ofpreservative solutions that typically are alcohol-based, and attempt todeposit the cells to preserve cell morphology.

There are several limitations to current cytologic preparation methods.These include distortions of cell morphology because of mechanicalforces involved in smearing or sedimenting the cells onto the slide. Thedeposition of cells onto the slide may result in cells overlapping oneanother, so that individual cell morphology cannot be visualized.Additional limitations associated with current cytologic preparationmethods include, although are not limited to, the following:inhomogeneous sampling of a specimen due to differential rates ofsedimenting cells onto a slide; the loss of cell clusters during certaintypes of preparation methods; the loss of small cells in methods thatdepend on density gradient methods of preparation; damage or nonspecificloss of inflammatory cells in centrifugation methods; and the inabilityto determine the absolute number of cell types in a sample, which may beimportant in determining the number of abnormal cells in a sample or incounting the number of inflammatory cells to determine the predominanttype of inflammation that is in a sample.

Examples of these limitations are found with the Pap testing techniquesthat may not adequately display certain cell clusters because ofsmearing or filtration processes used to prepare the slide. The densitygradient preparation method employed by the SurePath method may notcapture small, buoyant cells that float atop the gradient.Centrifugation methods may result in the inconsistent loss of smalllymphocytes, which can be problematic when an accurate differentialcount of inflammatory cells is required. Certain types of inflammatorylung diseases, such as idiopathic pulmonary fibrosis and sarcoidosis arecharacterized by unique profiles of inflammation that can only be usefuldiagnostically if an accurate enumeration of those cells can bedetermined.

A method of preparation of cytology samples that would not distortmorphology, and that would result in a homogeneous and quantifiablenumber of cells being placed on the slide would overcome the limitationsof current preparation methods.

Other examples involving body fluids include the detection of cells orcellular components that may be circulating in the peripheral blood. Forexample, cells from a non-hematological tumor may be found in the bloodand could be detected by visual examination or automated examination ofa slide. Special markers such as antibodies may be used to tag thesecells. Other cellular components such as specific proteins may also bepresent in the blood either intracellularly or extracellularly and couldbe evaluated on the slide, typically by using certain markers to tagthese slides. Certain inclusions in blood cells may also be detectable;for example parasites.

SUMMARY OF THE INVENTION

The present invention provides improved systems and methods forpreparing and applying cells from a body fluid on a slide. Additionallysystems and methods for imaging the cells are provided. The images maybe later used to perform tests including image-based counting andassessment of the morphology of the cells. The present invention may beused on a variety cells from body fluid including blood, bone marrow,urine, vaginal tissue, epithelial tissue, tumors, semen, spittle, andother fluids.

By way of example, in the case of analyzing blood or bone marrow,aspects of the present invention may used process a slide and optionallycapture an image of the slide. The image may be later used forperforming various tests that provide for a count of various cell types,or an assessment of the morphology of the cells. One example is acomplete blood count including image-based counting and assessment ofthe morphology of the formed elements of blood, including RBCs, WBCs,and platelets. Embodiments of the present invention may improve theaccuracy of the CBC as a result of direct visualization of the formedelements of blood. The use of the disclosed systems and processes forapplying a monolayer of cells onto a slide enables assessment of certaincell types, particularly of abnormal and immature WBCs that are found incases of abnormal bone marrow function including hematologicalmalignancies. Further, the present invention may decrease costsassociated with instrumentation; decrease cost of consumables andreagents; and require less operator time and reagents, fewer repeatedtests, and fewer moving parts. It may also reduce the turnaround timefor many of the CBC tests that currently require visualization of bloodcells after the instrumental portion of the test is completed, byallowing cells to be visualized on a monitor instead of under amicroscope.

Aspects of the present invention are effective at preserving cellmorphology. This may be important for patients with hematologicalmalignancies such as chronic lymphocytic leukemia (CLL) or acute myeloidleukemia (AML). The systems and processes for creating a monolayer ofcells from body fluid may enable detection of a larger number ofmorphologically well preserved blast cells and other immature or fragilecells. This would allow their more accurate recognition at an earlierstage of the leukemic or other disease process. Certain aspects of thepresent invention provide for preparing a substantially uniformdistribution of cells across a test area of a slide.

Aspects of this invention may relate to the application of cells frombody fluids to a slide and include possibly mixing the cells containedin the body fluid with a diluent, collecting a sub-sample (aliquot) of aknown volume from the solution, and then depositing the aliquot onto asubstratum such as a slide using a dispensing device or applicator. Thecells may be allowed to air dry or may be fixed (using a fixativesolution) or both, depending on the examination that is anticipated. Thecells may also be stained. The stained cells on the substratum may becounted and examined by an automated imaging system utilizing a computeror viewed by manual microscopic examination. Digital images may be shownon a computer display to reduce the need for manual microscopic review.

Aspects of the invention also relate to systems and methods forcollecting cells from a body site, placing the cells into a preservativesolution, mixing the cells in the solution to assure a homogeneousdistribution, collecting an aliquot of known volume from thepreservative solution and then depositing the aliquot onto a slide usingan applicator. The cells may be fixed, stained, or allowed to air dry,depending on the examination that is anticipated. The slide containingthe specimen may be used for either manual microscopic examination, orbe examined by an imaging technique that can enumerate the differenttypes of cells that are present on the slide.

Systems and methods of the present invention provide a number ofimprovements over prior art techniques. For example, an embodiment ofthe present invention may be used to determine the number of cells in asample of the cervix that are infected by the Human Papilloma Virus(this may indicate the viral burden, which is a prognostic factor toassess if an abnormality may progress, remain stable, or regress).Embodiments of the present invention may be able to determine how manyviral or infected cells are in the sample. Additionally, certainembodiments of the present invention may be able to determine thedifferential cell count in an non-gynecologic sample collected from abody cavity effusion. In further embodiments, the system or method coulddetermine that there is a large number of acute inflammatory cells in asample (which the system or method may use to determine the presence ofa bacterial infection). Similarly, if an embodiment of the presentinvention determined there were a high number of lymphocytes in aparticular sample this may suggest a viral infection, autoimmunedisease, or tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: is a perspective, schematic view of a system for analyzing bodyfluids.

FIG. 1B: is a perspective, schematic view of a system for analyzing bodyfluids.

FIG. 2: is a perspective view of a slide and slide holder.

FIG. 3: is an enlarged top view of the slide and slide specimen.

FIG. 4: is an alternate embodiment of the top view of the slide andslide specimen.

FIG. 5: is a graph illustrating the correlation between SYSMEX® brandhemology analyzer (hereafter “Sysmex”) RBC counts and the RBC countsgenerated using an embodiment of the invention.

FIG. 6: is a graph illustrating the correlation between Sysmex WBCcounts and the WBC counts generated using an embodiment of theinvention.

FIG. 7A: is a process flow schematic of the embodiment shown in FIG. 1A.

FIG. 7B: is a process flow schematic of the embodiment shown in FIG. 1B.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1A, a system 10 for analyzing body fluids isdisclosed. The system may comprise a platform 100, a light receivingdevice 200, a computer 300, an applicator 400, a gas circulation device500, a light source 600, a dispenser 800, a discharge device 900, aslide labeler 1000, and slide label reader 1100. The following sectionsbelow include capitalized headings intended to facilitate navigationthrough the specification, which are not intended to be limiting of theinvention in any manner.

The Platform 100

In embodiments that feature a platform 100, an advancer 110 may beconfigured to receive one or more slide apparatuses 700-700″. Theadvancer 110 may be attached to a surface, such as the top surface 101,of the platform. The advancer 110 may take the form of a belt as shownin FIG. 1A, the system may use a mechanical arm, gravity, magnetism,hydraulics, gears, or other locomotion techniques to move the slideapparatus along the surface 101 of the platform.

The platform 100 may also comprise a feeder 102 and a collector 106 forrespectively feeding and collecting the slide apparatuses 700 from or toa stack or rack. The feeder 102 may be equipped with a feeder propulsionmechanism 103 (such as rubberized wheels) for pushing the slides down aramp 104 onto the advancer 110. (Of course, embodiments of the inventioncould be built without a ramp, for example, if the feeder is level withadvancer 110, no ramp would be needed. Alternatively, a mechanical armcould be used to grab the slide apparatus 700 and place the slideapparatus 700 on the advancer directly.) Alternate mechanisms to propelthe slide out of the feeder 102 may be used such as magnets orhydraulics. The feeder may comprise a sensor for determining how manyslides are present. The sensor could measure the weight of the slideapparatuses 700 for example to determine how many slide apparatuses werepresent. FIG. 1A illustrates 3 slide apparatuses 700 stored in thefeeder 102. The collector 106 may also comprise a sensor for determininghow many slides are present in the collector 106. The sensor may informthe computer when a preset number of slides have been analyzed or mayinform the computer of the receipt of a slide on an ongoing basis.

The Light Receiving Device 200

The light receiving device 200 may be a microscope (such as brightfieldmicroscope), a video camera, a still camera, or other optical devicewhich receives light. The light receiving device may comprise anobjective, eyepiece, a stage or any combination thereof. In embodimentsusing a standard brightfield microscope, one containing an automatedstage (a slide mover 201) and focus may be selected. In one embodiment,a microscope may be attached to a motorized stage and a focus motorattachment. The microscope may have a motorized nosepiece, for allowingdifferent magnification lenses to be selected under computer 300control. A filter wheel may allow the computer 300 to automaticallyselect narrow band color filters in the light path. LED illumination maybe substituted for the filters, and use of LEDs may reduce the imageacquisition time as compared to the time required for filter wheelrotation. In LED and filter wheel embodiments, the light receivingdevice many contain an autofocus controller for shifting the focal pointof the light so that it is focused when it enters the light receivingdevice. For example, the autofocus controller may control the relativeposition of an objective or stage of the light receiving device 200 tofocus light on a lens of the light receiving device 200. A 1600×1200pixel firewire camera may be used to acquire the narrow band images.

In some cases, the light receiving device will receive light reflectedoff slide apparatus 700″ and store an image of that light. However,since the light emission source 600 can be positioned below theplatform, the light emission source may direct light so that it passesthrough the platform 100 and the slide 701 into the light receivingdevice 200. In some embodiments fluorescent emission from the cellularobjects may be detected in the light receiving device 200. The lightreceiving device may be connected to a computer through a link 11, andmay be capable of X, Y, and Z axial movement (in other embodiments amotorized stage or slide mover may provide X, Y, and Z movement.) Thelight receiving device may comprise a link 11 such as a wire as shown inFIG. 1A, or other wireless systems may be used. The light receivingdevice 200 and any of the other components may be interfaced with thecomputer 300 through a link (11-15) which may provide power to thecomponent, provide instructions from the computer 300 to the component,or allow the component to send information to the computer 300. Lightreceiving device 200 may contain pan, tilt, or locomotive actuators toallow the computer 300 to position the device 200 in an appropriateposition. The light receiving device may contain a lens that focuses thelight. The light receiving device may capture black and white or colorimages Alternatively, two or more light receiving devices could be usedto divide the processing time associated with capturing the images. Forexample, one light receiving device may operate as a low magnificationimage station while another light receiving device operates as a highmagnification image station. Similarly, in some embodiments, the system10, platform 100, computer 300, or light receiving device 200 may directa slide mover 201 to move the slide apparatus 700 in order to storeimages of all the cells in the slide. Using a slide mover 201 may bedesirable, if for example, the field size of the light receiving device200 is smaller than the specimen zone 710 (FIG. 3).

The Computer 300

The computer 300, may be a laptop as shown in FIG. 1A, or a server,workstation, or any other type of computing device. The computer maycomprise a processor, a display 320, an interface 310, and internalmemory and/or a disk drive. The computer 300 may also comprise softwarestored in the memory or on computer readable media such as an opticaldrive. The software may comprise instructions for causing the computerto operate the light receiving device 200, the applicator 400, theapplicator controller 490, the fan 500, the platform 100, advancer 110,light source 600, dispenser 450 or 800, or any component connected toone of these components. Similarly, the computer may receive informationfrom any of these components. For example, the software may control therate of dispersal of slides from the feeder 102, and feeder 102 mayinform the computer about the number of slides present. In addition, thecomputer 300 may also be responsible for performing the analysis of theimages captured by the light receiving device.

In an embodiment of the invention capable of preparing and analyzingcells from blood samples, the computer 300 may be able to calculate thenumber of a specific type of cell in a particular volume of blood, forexample for blood, red cell, white cell, and platelet counts and othermeasured and derived components of the CBC such as: hemoglobin content,red blood cell morphology, or WBC differential could be calculated. Theimage analysis software may analyze each individual field and sum thetotal red and white cell counts. To calculate the total counts permicroliter in the patient vial, the number counted on the slide ismultiplied by the dilution ratio and volume of the sub-sample. Resultsof the counts, morphologic measurements, and images of RBCs and WBCsfrom the slide may be shown on the display 320. In some embodiments, thecomputer 300 may be able to display numerical data, cell populationhistograms, scatterplots, and direct assessments of cellular morphologyusing images of blood cells displayed on the monitor. The ability todisplay cellular morphology provides users of the system 10, the abilityto quickly establish the presence or absence of abnormalities in cellmorphology that may warrant preparing an additional slide for manualreview by an experienced technician or other professional. The softwaremay provide the computer instructions to display images 331 receivedfrom the light receiving device or may cause the display 330 to show theresults 332 (in perhaps a chart or graph for example) of an analysis ofthe images. Similarly, the computer 300 may be able to enumerate thenumber of cells of a specific type in a particular blood volume orenumerate the number of damaged cells, cancerous cells, or lysed cellsin a particular volume of blood. The memory of the computer may containsoftware to allow the computer to perform the analysis process. Thecomputer may use one or more magnifications during the analysis. Whilethe example above describes using an embodiment of the invention forpreparing and analyzing cells from a sample of blood, embodiments of thepresent invention may be used for preparing and analyzing cells fromother fluids such as bone marrow, urine, vaginal tissue, epithelialtissue, tumors, semen, spittle, and/or other body fluids.

Although shown as one component, computer 300 may comprise multiplecomputers and a first computer could be used for controlling thecomponents and a second computer could be used for processing the imagesfrom the light receiving device 200. In some embodiments, the variouscomputers may be linked together to allow the computers to shareinformation. The computer 300 may also be connected to a network orlaboratory information system to allow the computer to send and receiveinformation to other computers.

The Applicator 400

In certain embodiments, the applicator 400 may comprise a syringe, amanual or motor driven pipettor or a motor controlled pump attachedthrough a tube to the applicator tip 405. While many different types ofpipettes or syringes could be used, test results have shown improvedresults can be obtained through using an applicator 400 having betterthan 2% accuracy. The pump may be a peristaltic pump, a syringe pump, orother similar device that allows small volumes of fluid samplescontaining cells to be aspirated and dispensed through an orifice.Typically such an orifice will be contained in a tip 405 that is two tofive millimeters in outside diameter with an inner diameter of 0.5millimeters. The tip 405 may be disposable or washable. The tip 405 maybe rounded to facilitate insertion and cleaning of the tip. Fluid flowthrough the tip is controlled to allow a thin layer of body fluidcontaining cells to be deposited onto the slide. By optimizing flow ratethrough the tip and the relative speed and height of the tip over theslide an appropriate density of cells can be deposited onto the slide.Each of these factors influences the other, so the proper combination ofheight, flow rate through the tip, and speed over the slide must bedetermined. In one embodiment the flow rate through the tip is 0.1microliters per second while the tip is moving at a speed of 30millimeters per second over the slide surface at a height of about 70microns. In another embodiment, for example when the body fluidcomprises undiluted blood, the flow rate through the tip isapproximately 0.04 microliters per second while the tip is moving at aspeed relative to a point on the slide of 50 millimeters per second at aheight of about 10 microns above the slide surface. The viscosity andconsistency of the particular body fluid specimen will influence theflow rate through the tip and the relative speed and height of the tipover slide required to ensure that an appropriate density of cells aredeposited on the slide for examination.

In use, the applicator 400 may comprise a known volume of body fluidsuch as 30 microliters (ul). Some body fluids may need to bepre-processed to disperse cells that may be clumped together or tominimize mucous or other protein material that may cause the cells tostick together. Other body fluids such as urine may need to concentratedbefore the body fluid is placed into the applicator. The applicator maymix this fluid with a stain or diluent, and eject a portion of thisfluid onto the slide apparatus 700 (particularly the specimen zone 710,FIG. 3). A typical sub-sample would be an aliquot of approximately ½ μlto 2 μl, but may be in the range of 1/10 to 10 μl. In some embodiments,the system 10 or applicator 400 may contain a first reservoir 420 forstoring the body fluid and a second reservoir 430 for storing diluent.In some embodiments the body fluid will not be diluted.

The system 10 or applicator 400 may contain one or more dispensers 800.The dispenser 800 (or 450 in FIG. 1B) may be used to direct a fixativeor a stain onto the slide 701. In this embodiment, the applicator 400may contain one or more fluid chambers 410 to eject body fluid, diluent,stain, and fixative from the applicator 400. Some dispensers may be ableto store both fixative and stain, and direct them sequentially onto theslide, or in alternate embodiments two dispensers may be used (one forthe fixative and one for the stain.) Excess stain and fixative may beremoved from the slide, by tilting the slide apparatus so that it isorthogonal (or angled) to the platform surface 101. A slide tilter 801may be used for this purpose. Slide tilter may comprise a simple wedgeas shown, or may comprise a mechanical arm to tilt the slide.

In the embodiment shown in FIG. 1A, the stain dispenser is attached tothe platform 100. Examples of stains compatible with embodiment shown inFIG. 1A may include: Wright-Giemsa stain, Geimsa stains, and Romanowskystains. Other solutions that could be dispensed are fixatives (methanol)and buffer solutions. Other visualization methods involvingimmunocytochemical reagents or other markers of specific cell componentsmay also be used. The stain dispenser may also be embodied as a stainreservoir 450 and attached to the applicator 400 (see FIG. 1B). Examplesof stains compatible with the embodiment shown in FIG. 1B may include:Romanowsky stains, reticulocyte stains, and stains using specificantibodies. Additional stains may be used with embodiments of theinvention including hematoxylin and eosin; immunocytochemical stains;histochemical stains for viewing cellular components; and antibody,aptamer or other stains based on binding a ligand to an antigen. In theembodiment having dispenser 800, the dispenser can dispense stain ontothe slide apparatus (particularly the specimen zone 710.) Dispenser 800may take the form of a peristaltic pump. In the embodiment having astain reservoir 450, the stain may be mixed in with the body fluid andthe diluent from reservoirs 420 and 430. The body fluid and the diluentmay be mixed together by a mixer 440, which can mix the fluid anddiluent in certain ratios. In an alternate embodiment, the slide couldbe immersed into one or more baths of the fixing and staining solutions.In another embodiment, fixing and staining solutions could be movedacross the slide using capillary action.

Various fixatives and diluents may be used with the present invention.For example 85% methanol can be used as the fixative. For some stains anethyl alcohol or formaldehyde based fixative might be used. Diluentsuseful for diluting whole blood for example, may include salt solutionsor protein solutions. Salt solutions range from “physiological saline”(0.9N), to complex mixtures of salts, to the commercial preparationPLASMALYTE® that simulates virtually all the salts found in human bloodserum. Protein solutions can range from simple solutions of bovinealbumin to PLASMANATE®, a commercial preparation with selected humanplasma proteins. Such preparations can vary in protein concentrations,buffers, pH, osmolarity, osmalality, buffering capacity, and additivesof various types. Synthetic or “substitute” versions of these solutionsmay also be usable, including FICOLL® or Dextran or otherpolysaccharides. Other substitutes may be used. An example of a diluentis PLASMALYTE® plus PLASMANATE® in the proportion of 4:1 (PlasmanatePLASMALYTE®:PLASMANATE®). Another example of a diluent is 5% albumin.When analyzing whole blood, a dilution of 2 parts blood to 1 partdiluent can be used, where the diluent is a physiologically compatiblesolution, but a range of dilution from 0:1 (no dilution) to 10:1(diluent:blood) may be used in alternate embodiments.

Embodiments of the present invention may also be used with body fluidsamples that require concentration before applying flows of cells fromsuch fluid samples to a slide. For example, body fluids such as urinemay require concentration to ensure that the flows of cells placed onthe slide contain sufficient quantities of cells onto the slide foranalysis. Fluid samples may be concentrated through techniques such ascentrifugation, filtration, or use of cell concentration tubes.

The applicator may comprise a hydraulic piston for pushing the fluid outof fluid chamber 410 (like a syringe or a pipette). A tip 405 may beprovided for adjusting the flow rate of the fluid. While size of the tipdoes not affect the speed (ul/sec) in which the solution flows out ofthe tip, generally, the smaller the opening in the tip, the greater theforce generated by the fluid flowing from the tip. Additionally, thesize of the tip affects thickness of the fluid flows 750 shown in FIGS.2 and 3. A tip having a 0.3 millimeter inner diameter may provide for aflow rate of 0.1 microliters per second, and the distance from a middlepoint 751 of the first flow to the middle point 752 of the second flowmay be 500 microns. In order to create the flows 750 shown in FIGS. 2and 3, the system 10 may be configured to account for the variances inthe number of cells in a given body fluid specimen. For example, inhuman peripheral blood samples, the range is large but within one orderof magnitude. In order to accurately count the blood cells, the overlapbetween red blood cells should be minimized. One method to provideminimal overlapping between cells is to lay down non-touching rows ofcells from the tip of the applicator. Increasing viscosity of thediluted fluid or the type or amount of diluent may affect the width ofthe final settlement positions of the flows 750. By selecting a distancebetween rows to allow for the typical variation in blood samples, allcells can be counted in all samples. For many samples these gaps will beseen between the flows; however this does not affect the image analysisand the row and gap effect tends not to be noticed during highmagnification manual review under the microscope. To avoid these gaps, alight receiving device could be attached to the applicator or positionednear station A (see FIG. 7A) to allow the computer 300 to determine thewidth of the first flow 751 (FIG. 3) formed by directing the cells ontothe slide. By determining the width of the flow, i.e. how far the bloodflows sideways from location the fluid was placed on the slide, thecomputer 300 could cause the applicator to adjust the gap size betweenthe flows. The computer 300 calculates the distance the second flow 752(FIG. 3) needs to be from the first flow 751, and place the flows sothat they settle adjacent to one another minimizing the formation of anygaps between the flows. Using this process, a gapless or contiguous flowof cells can be applied to the specimen zone 710.

To physically place the cells on the slide 701, the computer 300 coulddirect the applicator controller 490 to perform the body fluidapplication process 7B (see FIG. 7B) which involves moving the bodyfluid chamber 410 in the X, Y, or Z directions to position the tip 405so that it is tracing the eventual locations of the flows 750. In someembodiments, two of the directions may be held fixed, so that the tip405 moves in a relatively straight path in relation to the slide 701. Inother embodiments, one of the directions may be held fixed, so that thetip moves in a wavy, arcuate, or circular path in relation to the slide701. In still other embodiments all three directions may be modified asthe tip 405 is moved in relation to the slide 701. In some embodiments,the X, Y, and Z directions are all perpendicular to each other affordingthe applicator the ability to move in any direction in a threedimensional coordinate system. In other embodiments, the system movesthe slide 701 in the X, Y, or Z directions to position the slide underthe tip 405 and move the slide while the tip 405 applies flows of cellsfrom body fluid on the slide.

The computer 300 may be connected to the applicator controller 490 tocontrol this movement. In the embodiment shown in FIG. 3, the controllermay position the tip at the top left corner of the specimen zone 710 andproceed to place fluid sample onto the slide by dispensing the fluidfrom the fluid chamber 410. As the tip dispenses fluid, the controller490 may move the tip in the positive X direction to the top rightportion of the specimen zone 710 (see FIG. 3). Once the top rightsection is reached, the controller 490 may move the tip in the negativeY direction one flow width. The flow 750 width may range from 300 to1000 microns, and flow thickness increases as the flow rate of fluid outof the tip increases and/or the speed of the tip across the slidedecreases. Additionally the viscosity of the fluid and diluent choicemay affect the width of the flow 750 (FIG. 3). Typically, the cells ofthe fluid will settle within a few seconds once placed on the slide.Once the tip has been moved one flow width, the controller may move thetip in the negative X direction to the leftmost side of the specimenzone 710. Once the leftmost side is reached, the tip again may be movedone flow width in the negative Y direction. This process may be repeateduntil the entire specimen zone is covered or until a specific quantifyof body fluid has been dispensed on the slide such as one microliter. Inalternate embodiments, the diluted body fluid could be applied to slidewith a fixed applicator and slide which moves via the moveable slidecontroller 760 (this application process 7A is shown on FIG. 7A.) Theslide controller 760 may be moveable in the X, Y, Z direction to movethe slide apparatus in similar positions to allow the applicator toplace flows 750 of body fluid on the specimen zone 710.

The number of cells placed on the slide 701 using this method will varydepending on the type of body fluid being examined and the dilutionratio. Assuming whole blood were being analyzed with a 1:3 ratio(blood:diluent), about 900,000 red blood cells, 45,000 platelets, and1,000 white blood cells would be placed on the slide. Though FIG. 3shows the generation of a uniformly distributed fluid specimen in arectangular shape, other shapes may be constructed in a similar manner.FIG. 4, shows for example, a fluid flow comprising a plurality ofconcentric circles. Like FIG. 3, the fluid flows 750 are placed adjacentto one another to create a uniform viewing field. This process providesa highly uniform distribution of cells across the specimen zone 710,facilitating the analysis process. Additionally, the computer 300 canalter the appearance and width of the fluid on the zone 710. Forexample, the computer 300 may control the speed at which the tip movesacross the specimen zone, which would affect the thickness of the fluidresting on the zone. In some embodiments, speeds of 10 to 100 mm/s maybe selected in order to provide the zone with a specimen which is aboutone cell thick. The controller 490 also may select the height of the tipabove the slide 700. A height of 70 +/−40 microns above the slide may beused in order to minimize damage to fluid cells when they come intocontact with the slide apparatus 700, and to maintain fluid flow fromthe tip to the substrate.

The Gas Movement Device 500

Gas movement device 500 may comprise a fan (such as shown in FIG. 1) ormay comprise other gas movement devices such as a compressor or a bellowfor example. Gas movement device 500 may be connected directly to thecomputer 300 or may be connected through another component such as theplatform 100 or the applicator 400 (as shown.) The gas movement devicepushes gas (in some cases atmospheric air) across the slide to controlthe rate at which the slide dries. Moving too much air too quickly (i.e.too high of a fan speed) across the slide can cause cells in thespecimen to burst due to excessively rapid drying, and too little airtoo slowly (i.e. too low of a fan speed) across the slide can cause thecells to dry too slowly and appear to shrink. The computer 300 mayselect the amount of air that moves across the slide in a period of time(i.e. the cubic feet of air per second) based upon the distance the gasmovement device is from the slide, the type of fluid being analyzed, therelative humidity, the width of the flows, and averages thickness of theflows (this would be the amount of cells in each flow in the Zdirection). The gas movement device 500 may be placed near the slideapparatus 700, and positioned so that the device directs gas to strikethe slide at an angle of 30-60° angle (45° degrees can be used) for aperiod of about 15 to 20 seconds. In some embodiments, the computer cancontrol of humidity and temperature settings in the vicinity of thesystem to allow the drying process to occur without the use of a gasmovement device 500.

The Light Emission Device 600

Two different embodiments of light emission device 600 are illustrated.In FIG. 1A, light emission device 600 comprises a housing 601, amultispectrum light source 610, a number of light filters 620, 620′, and620″, and a filter selector 621. As shown in FIG. 1A, a portion of thehousing has been removed to better show the light source 610. Lightsource 610 may comprise a white light source or other multispectrumlight source such as a halogen bulb, florescent bulb, or incandescentbulb etc. Filters 620-620″ may be used to filter the multispectrum lightinto a single wavelength or a narrow band of wavelengths. The filterselector 621 may select which filters appear in front of the lightsource 610. In some embodiments more than one filter may be used toallow a particular range of light to illuminate the slide. Filterselector 621, may comprise a rotation motor and a rod to spin thefilters in and out of the path of the light. In a second embodimentlight source may comprise one or more lasers or LEDs (630) which emit anarrow band of light (see FIG. 1B). An advantage for using LEDs in thissystem 10, is that LEDs can rapidly be switched on and off, allowing thelight receiving device, for example a single black and white camera, toacquire the multiple spectral images in a very short time. LEDs alsoproduce narrow bandwidths of illumination, typically from 15 to 30 nmfull width at half maximum (the breadth of the wavelength intensitydistribution at half of the peak brightness of the maximum intensity).Also, LEDs in the visible range do not project heat-producing infraredenergy into the optical system and are relatively long lived as comparedto conventional lamps. An advantage of using narrow-band illuminationrather than unfiltered white light (i.e. broad-band illumination) isthat using narrow band illumination increases the sharpness of theimages generated by the light receiving device 200. If the lightreceiving device 200 contains a lens, the presence of the lens may causesome chromatic aberration that results in slight focus shifts or imagequality degradation when using different colors. With white lightillumination this can result in an overall degradation of the imagequality. The light receiving device 200 may capture a black and whiteimage for each narrow-band of illumination. The computer 300 may correctfocus and image quality for each wavelength by adjusting the focaldistance or the distance of the lens from the slide. In someembodiments, the computer 300 may shift the focus position of the lenswhile a number of light colors are emitted sequentially to improve thequality of the image.

Various wavelengths of light may be directed by the light emissiondevice 600. Two to eight or more different wavelengths of light may bedirected at the slide apparatus 700. For example, wavelengths ofapproximately 405-430 nm are useful for imaging a hemoglobin-only imagefor assessing RBC morphology and hemoglobin content. Using an imagetaken with such a wavelength designed to show only red blood cells mayalso show red blood cells that are touching white blood cells. Thetouching red blood cells may be digitally removed from images to make iteasier for the computer to detect the white blood cell borders in orderto make more accurate cellular measurements and enumeration. Lightemitted at 570 nm may be useful to provide high contrast images forplatelets and nuclei. Other wavelengths may be chosen in order to bestdiscriminate the colors of basophils, monocytes, lymphocytes (all shadesof blue), eosinophils (red), and neutrophils (neutral color). Forcounting platelets, for example, two colors of illumination may be used(such as 430 nm and 570 nm). A high contrast image may be obtained bysubtracting the 430 nm image from the 570 nm image. Light having awavelength of 430, 500, 525 or 600 is particularly effective at showingcell color information, although the light emission device may use lightat wavelengths between 400 nm and 700 nm inclusive. These wavelengthswill also be used for the display of the color images if appropriate.Otherwise one or two additional images may need to be taken for the 200+cells that will be analyzed for the differential count and which may beshown on the display 320. Typically the narrow-band images will bechosen from the range of 400 nm to 750 nm. Test results have shown thattwo to eight separate light colors to work well, with three to fourseparate light colors being optimal. The computer 300 may be able tofurther refine the images by compensating for spatial shifts. Also thecomputer may combine the various colored images to generate multi colorimages for display or analysis. Numeric descriptors of the individualimages or combined images can be used to determine spatial,densitometric, colorimetric and texture features of the cells forclassification of the cell types. A further advantage of using narrowband illumination is that narrow band illumination allows for theelimination of the use of oil objectives or coverslips. Light isrefracted when the light passes from glass to air. Prior art systemshave used oil objectives or coverslips to minimize this refraction atair to glass transitions, but having to add oil or coverslips adds stepsto processing the slides, and increases the per slide preparation timeand analysis cost. To overcome this deficiency of the prior art systems,a combination of narrow band LEDS or filtered light can be used withoutthe need to use coverslips or oil. Reducing the variance or bandwidth inthe wavelengths of the light decreases the distortion in the imagecaptured by the light receiving device 200 when the light passes throughthe slide 701. The computer 300 may also instruct the light emissiondevice 600, to focus the light from the light source (either 610 or 630)so that the light is properly focuses on the slide. To do this, thecomputer 300 may instruct a focus adjustor to optimize the focus foreach color of light.

The Slide Apparatus 700

FIGS. 1A, 2, and 3 illustrate an embodiment of the slide apparatus 700comprising a slide 701, a specimen zone 710, a slide frame 720, and aslide holder 730. However, other embodiments of the invention may notrequire the use of a slide holder 730 or slide frame 720. Additionallythe specimen zone 710 boundary mark is optional as well, and maycomprise one or more hydrophobic rings or other painted marks. Theserings may help contain the blood sample, and also make reviewing imagesof the slides easier by quickly locating the specimen zone when a slideis viewed manually under a microscope (the may also assist the analysisprocess in interpreting the image.) The rings may also assist infacilitating the transfer of the stain onto the slides. Additionally,while the specimen zone has been illustrated as a rectangle other shapessuch as a circle or triangle may be used. Different size specimen zonesmay be used, including zones having a total area of one half to threesquare centimeters. The slide 701 may be manufactured from glass orplastic and may be 1 inch tall by 3 inches wide by 1 mm thick. Alsoshown on FIGS. 2 and 3 is a fluid sample dispersed on the slide in flows750. The fluid can be dispersed in flows as shown in FIG. 3, or in aspiral pattern as shown in FIG. 4.

The Discharge Device 900

With reference to FIG. 1B, the system may comprises a discharge device900 for pretreating the slide 701. The discharge device may take theform of a corona discharge device. The discharge device 900 may cleanthe slide 701 by creating a high intensity heat to burn off smallparticles to clean the slide to create a hydrophilic surface.Electro-Technic Products, Sawicki, Pa., makes a corona discharge devicecompatible with embodiments of the present invention. To perform thepretreatment, the computer 300 would turn on the discharge device 900,and cause the slide apparatus controller 760 to move the slide in aspiral or raster motion for about 15 seconds (though a range of 1-20seconds could be used). The discharge device may be set at an angle fromthe slide, or may be positioned directly above the slide. Typically, thedischarge device 900 may be positioned approximately 10 to 20 mm abovethe slide.

The Slide Labeler 1000 and Slide Label Reader 1100

The system 10 may optionally include a slide labeler 1000 and optionallya slide label reader 1100. The slide label reader 1000 may be situatedon the platform 100 near the feeder 102 as shown in FIGS. 1A and 1B ormay be free standing or attached to other components. Slide labeler 1000may place a label on the slide. A label 770 may include items such asstickers, barcodes, RFID tags, EAS tags, or other type of markings onthe slide. FIG. 3 shows an exemplary slide having a UPC bar code labelon it, but other markings conventions may be used. Moreover, themarkings may be applied directly to the slide via paint or ink, or maythey may be stuck to the slide using a writing medium and an adhesive(for example, a sticker).

The system 10 may comprise a slide label reader 1100. Slide label reader1100 may read markings placed on the slide from the slide labeler 1000or by labelers external to the system. The slide label reader 1100 couldcomprise an interrogator, a bar code reader, or other optical device. Insome embodiments, the system 10 may be able to determine informationfrom the labels 770 without a slide label reader 1100 by using the lightreceiving device 200 to capture an image of the label 770. The computer300 or the light receiving device (if it contains a processor andmemory) could perform image processing on the image containing the labeland determine the information about the label 770.

Bone Marrow

As discussed above, the present invention may be used to analyzeperipheral or whole blood. The invention can also be used, however, tostudy cells of various types of fluids 590 comprising bone marrow,urine, vaginal tissue, epithelial tissue, tumors, semen, spittle, andother body fluids For example, the preparation methods and analysistechniques described here can also be applied to bone marrow aspirationsamples. Bone marrow samples have a higher cellular density and containmany immature red and white blood cell types that are seldom found inperipheral blood. The technique of preparing a thin layer of cells,staining with a Romanowsky stain and analyzing with image analysis canbe applied to bone marrow aspirates as well, however more sophisticatedimage analysis may be needed to discriminate the additional types ofcells.

As with peripheral blood samples, bone marrow samples may be collectedinto a container with an anticoagulant. This anticoagulant may be EDTAor heparin. Additional diluting or preserving fluid may be added to thesample. In the instrument described here a bone marrow sample would beprepared by first agitating the sample to provide a thorough mixing. Dueto the uncertain cellular density of such samples one or more dilutionsmay be prepared and pipetted onto the slide or slides. In oneembodiment, a triple dilution process may be used to create threespecimens. A first specimen may be created by adding 2 parts diluent toone part bone marrow. The first specimen may then be dispensed onto afirst portion of the specimen zone 710 of the slide 701. A secondspecimen may be created by adding four parts diluent to the bone marrow.The second specimen may then be dispensed onto a second portion of thespecimen zone 710 of the slide 701. A third specimen may be created byadding eight parts of diluent to the marrow. The third may then bedispensed onto a third portion of the specimen zone 710 of the slide701.

For viscous body fluids, including but not limited to bone marrow, itmay be desirable to provide the system 10 with a serial dilutionmechanism (?). In one embodiment of this process, the applicator 400 candirect one or more flows of cells onto a slide 701. The light receivingdevice 200 can capture an image of the flows of cells, and the computer300 can determine whether the flows are sufficiently dilute for forminga monolayer of cells on the slide, performing an accurate count of aparticular cell type, or capturing images that allow for an assessmentof cellular morphology. If the flows are not sufficiently dilute, thecomputer can instruct the mixer 440 to further dilute the body fluid.The applicator 400 can then apply a more dilute flow of cells to theslide 701, which the light receiving device 200 can image. The computer300 can again determine whether the flows are sufficiently dilute forforming a monolayer, counting, or assessining morphololgy and if not,the system 10 can instruct the mixer 440 to further dilute the bodyfluid. This process can be repeated until a sufficiently dilute bodyfluid sample is created. In some embodiments, the applicator 400 willapply flows of cells along the entire slide before the light receivingdevice 200 images the cells. In other embodiments, a subset of cellsflows (e.g. 3-10) may be applied before the cells flows are imaged. Insome embodiments, the computer 300 can analyze the captured image of theflow of cells, and determine an approximate amount of diluent necessaryto dilute the body fluid so that subsequent flows of cells may becounted when they are imaged. In other embodiments, the system 10 maycontain preset dilution intervals to as apply if the system determinesthe current dilution ratio is not sufficient (i.e. if the flows are notsufficiently dilute, try 1:1 (diluent:body fluid). If 1:1 is too low andthe flows are still not sufficiently dilute, try 2:1 (diluent:bodyfluid), etc.) In some embodiments, a user of the system can select via auser input specific dilution intervals for the body fluid such as nodiluent, 3:2 (dilutent:body fluid); 4:1; or 8:1. In some embodiments,the applicator 400 may place a first group of flows of cells onto aslide; a second group of flows of cells onto the slide; a third group offlows of cells onto the slide, etc; wherein each group of flows of cellshas a different diluent to body fluid ratio. Alternatively, theapplicator 400 may apply a first group of flows of cells onto a firstslide; a second group of flows of cells onto a second slide; a thirdgroup of flows of cells onto a third slide, etc; wherein each group offlows of cells has a different diluent to body fluid ratio. Finally,while the above serial dilution process is contemplated to be especiallyuseful for viscous body fluids such as bone marrow, this process may beused for less viscous body fluids such as peripheral blood or semen aswell.

For the image analysis, a low magnification assessment of the cellulararea on the slide could chose the optimum one third for subsequentanalysis. Once the proper area of the slide is selected, 200+ bonemarrow cells would be measured to determine the differential count.

Reticulocytes

The system 10 may also count the number of reticulocytes in a bloodsample. Using a Romanowsky stain to mark RNA, the computer 300 can countthe number of reticulocytes present in the specimen. When a Romanowskystain is used, the reticulocytes appear slightly bluer than other redblood cells, and are usually slightly larger. The computer 300 can useits analysis process (16A or 17B, of FIGS. 7A and 7B) to quantify theblue component of the red cells. The analysis process could measureintegrated optical density of a cell's difference image created bysubtracting one image taken with blue light of 430 nm (range of 400 to470 nm) from an image taken with non-blue light of 600 nm (range of 470to 750 nm). The analysis process could correlate the number of red bloodcells with a defined range of integrated blue component to a number ofreticulocytes counted manually or by flow methods using special stains.The accuracy of the analysis process can be further improved byrequiring the analysis process (16A or 17B) to measure the size, shape,color, and measured characteristics of cellular objects. For example,the analysis process could detect the difference between a red bloodcell with a bluish platelet lying under or over a red blood cell asopposed to a true reticulocyte. In other embodiments, a special stainmay be used to mark RNA in the cells, and an imaging method could beused to detect the presence of this stain.

Process Flows

Embodiments of the present invention are contemplated to processmultiple slide apparatuses 700 in a pipelined series as shown in FIG. 1Aor 1B, but embodiments which process the slide apparatuses 700 inparallel may also be constructed. Embodiments may be constructed whichcan process a large number (e.g. 10-20) of slide apparatuses in seriesor in parallel, or smaller volume systems 10 can be constructed(processing 4-8 slides at a time.) The following two paragraphs describean example process flow for FIGS. 1A and 1B, but alternate process flowsare possible and feasible through alternate embodiments of theinvention. These process flows are also illustrated in FIG.7A and 7B.Additionally, other configurations of the system are possible, and wouldlikely have different process flows. Moreover, although the steps arepresented in a series, many of the steps may be presented in a differentorder or performed simultaneously. Finally, most of the following stepsare optional, and may be removed from the process flow.

In the embodiment shown in FIGS. 1A and 7A, the software stored in thememory of the computer 300 may cause the computer to control the order,speed, and variables associated with processes 1A-16A. The process maybegin with computer 300 sending an instruction to the slide labeler 1000to place a label 770 on the slide 701. The labeling process 1A, may beperformed in the feeder 102 or may be performed on the ramp 104 or atthe slide apparatus controller 760. To move the slide apparatus 700 fromthe feeder 102, the computer 300 may send an instruction to the feeder102 to activate the feeder propulsion mechanism 103. The computer 300may also cause a feeder process 2A to begin which may include moving theslide apparatus 700 onto the advancer 110. The feeder process mayinclude utilizing the sensor to determine how many slides are in thefeeder 102. The computer may cause the advancer 110 to initiate anadvancing process 3A including moving the slide to the applicationstation A, and onto the slide apparatus controller 760 (if one ispresent). Once the slide apparatus is on the slide apparatus controller,the slide may be pretreated by the discharge device 900. Thepretreatment process 4A may include the slide controller 760 rotating orspinning the slide apparatus 700 as the discharge device burns thedebris from the slide 701. Once the optional pretreatment process 4A iscompleted the applicator process 5A may begin. The applicator process 5Amay comprise having an operator fill the reservoir tank 420 with diluentand reservoir tank 430 with body fluid. Body fluids such as urine,vaginal tissue, epithelial tissue, tumors, semen, spittle, peripheralblood, bone marrow aspirate or other body fluids may be used.Alternatively, the fluids may be aspirated automatically from apatient's sample vial. The mixer 440 may begin the mixing process 6A tomix the diluent with the body fluid in a certain ratio such as 2:1 (bodyfluid:diluent) to form a diluted body fluid. To apply the diluted bodyfluid to the slide 701, one of two body fluid application processes 7Aor 7B (FIGS. 7A and 7B, described above in conjunction with theapplicator 400) may be performed (but either process could be used forboth embodiments). After the application completes, the advancer 110 maycontinue the advancing process moving the slide apparatus to a secondstation B. Once the body fluid application process 7A is completed, thedrying process 8A may begin. The drying process may include using thegas movement device 500 to direct gas onto the slide for a period oftime (such as 20-30 seconds). Once the slide is dried, the body fluidmay be fixed using the fixation process 9A. After the body fluid isfixed, it may be stained using the staining process 7A. After the bodyfluid is stained, the excess stain may be removed using a stain removingprocess 11A. The stain removing process 11A may include a slide tiltingprocess wherein the slide is tilted at least partially in order to allowthe stain and or fixative to drain off the slide. To capture images ofthe specimen, the advancer 110 may continue advancing the slideapparatus to the imaging station C. At the imaging station C, the systemmay activate specimen illuminating process 12A and an imaging process15A, which uses the light emission device 600 and light receiving device200 respectively to illuminate the specimen and to capture images of theilluminated specimen. The computer 300 may direct the light emissiondevice to apply to different filters to the light to change thewavelength of emitted light using the light filtration process 13A.Alternatively, the LED illumination process of 13B may emit one or morewavelengths of light if a light emission device 600 comprises one ormore LEDs. A slide movement process 14A may be performed by the slidemover 201 to position the slide 701 in various X, Y, Z directionalpositions. Since in many embodiments, the magnification of the lens ofthe light receiving device will generate a view field that only containsa part of the total area of the specimen, the slide movement process 14Amay be utilized to move the specimen into different X, Y positionsallowing the light receiving device 200 to take multiple images 331 tocapture the entire specimen. The slide mover may also be able to movethe slide to multiple imaging stations allow light receiving devices totake images at various magnifications. The slide mover may also be ableto move the slide in the Z direction allowing one or more lightreceiving devices to take images at various magnifications, focusposition and light wavelengths. The system 10 may use the label reader1100 to read the labels on the slides (using the label reading process16A), or alternatively the computer may recognize symbols on the labelusing image recognition software. The light receiving device maytransfer the images to the computer through link 11. The computer maysave the images in internal memory and use its software to analyze theimages (using the analysis process 16A ) to count the cells and performcalculations on the resulting data. The software may generate resultsincluding tables, charts, or a graph of the results 332, and may displaythe images 331 on the display 320 of the computer 300.

A second process flow is shown in FIG. 7B (also refer to FIG. 1B). Theprocess may begin with computer 300 sending an instruction to the slidelabeler 1000 to place a label 770 on the slide 701. The labeling process1B, may be performed in the feeder 102 or may be performed on the ramp104 or at the slide apparatus controller 760. To move the slideapparatus 700 from the feeder 102, the computer 300 may send aninstruction to the feeder 102 to activate the feeder propulsionmechanism 103. The computer 300 may also cause a feeder process 2B tobegin which may include moving the slide apparatus 700 down the ramp 104onto the advancer 110. The feeder process 2B may include utilizing thesensor to determine how many slides are in the feeder 102. The computermay cause the advancer 110 to initiate an advancing process 3B includingmoving the slide to the application station A, and onto the slideapparatus controller 760 (if one is present). Once the slide apparatusis on the slide apparatus controller 760, the slide 701 may bepretreated by the discharge device 900. The pretreatment process 4B mayinclude the slide controller 760 rotating or spinning the slideapparatus 700 as the discharge device burns off any debris on the slide701. Once the optional pretreatment process 4B is completed theapplicator process 5B may begin. The applicator process 5B may comprisehaving an operator fill the reservoir tank 420 with diluent andreservoir tank 430 with body fluid. Alternatively, the fluids may beaspirated automatically from a patient's sample vial. Body fluids suchas peripheral blood or bone marrow aspirate may be used, although fluidscomprising bone marrow, urine, vaginal tissue, epithelial tissue,tumors, semen, spittle, and other body fluids may be prepared andanalyzed using embodiments of the invention. The applicator 400 maycontain a third reservoir for containing the stain, and perhaps a fourthreservoir for containing fixative (however, in other embodiments thestain and fixative could be stored in the same reservoir). The mixer 440may begin the mixing process 6B to mix the diluent with the body fluid(and possibly the stain and fixative) in a certain ratio such as 2:1(body fluid:diluent) to form a diluted body fluid. In these embodiments,the applicator 400 would apply the stain and or the fixative after thebody fluid is applied to the slide using the staining process andfixative process respectively. Once the slide is dried, the body fluidmay be fixed using the fixation process 9B. After the body fluid isfixed, it may be stained using the staining process 7B. To apply thediluted body fluid to the slide 701, one of two body fluid applicationprocesses 7A or 7B (described above in conjunction with the applicator400) may be performed (but either process could be used for bothembodiments). Once the body fluid application process 7B is completed,the drying process 8B may begin. The drying process may include usingthe gas movement device 500 to direct gas onto the slide for a period oftime (such as 20-30 seconds). After the body fluid is stained and fixed,the stain may be removing using a stain removing process 11B. The stainremoving process 11B may include a slide tilting process wherein theslide is tilted at least partially in order to allow the stain and orfixative to drain off the slide. To capture images of the specimen, theadvancer 110 may continue advancing the slide apparatus to the imagingstation C. At the imaging station C, the system may activate specimenilluminating process 12B and an imaging process 15B, which uses thelight emission device 600 and light receiving device 200 respectively toilluminate the specimen and to capture images of the illuminatedspecimen. The computer 300 may direct the light source 600 to apply asequence of narrow band light onto the slide 701 using LED illuminationprocess 13B. Alternatively, if a light emission device 600 with filtersis provided, the computer 300 may direct the light emission device toradiate light and apply different filters to the light to changewavelength of emitted light using a light filtration process 13A. Onceslides are illuminated, a slide movement process 14B may be performed bythe slide mover 201 to position the slide 701 in various X, Y, Zpositions. Since in many embodiments, the magnification of the lens ofthe light receiving device will generate a view field which onlycontains a part of the total area of the specimen, the slide movementprocess 14B may be utilized to move the specimen into different X, Ypositions allowing the light receiving device 200 to take multipleimages to capture the entire specimen. The slide mover may also be ableto move the slide to multiple imaging stations that allow lightreceiving devices to take images at various magnifications. The slidemover may also be able to move the slide in the Z direction to allow thelight receiving device to take images at various magnifications. Thesystem 10 may use the label reader 1100 to read the labels on the slides(using the label reading process 16B), or alternatively the computer mayrecognize symbols on the label using image recognition software. Thelight receiving device may transfer the images to the computer throughlink 11. The computer may save the images in internal memory and use itssoftware to analyze the images (using the analysis process 17B) to countthe cells and perform calculations on the resulting data. The softwaremay generate results including tables, charts, or graph of the results,and may display the images 331 or the results 332 on the display 320 ofthe computer 300.

Test Results

To determine the accuracy of this method, computer algorithms weredeveloped to count RBCs and WBCs from digital images taken from a fluidsample comprising blood.

Table 1 below shows a summary of data for 34 slides. “Invention” datarepresents red and white blood cell counts from slides produced usingthe method described above, and analyzed using image analysis countingalgorithms. “Sysmex” data represents red and white blood cell countsfrom a commercial “flow-based” automated CBC analyzer. Note that thespecimens include very high and very low red blood cell counts and whiteblood cell counts, respectively.

TABLE 1 Invention Sysmex Invention Sysmex Count Count Count CountSpecimen RBC × 10⁶ RBC × 10⁶ WBC × 10³ WBC × 10³ 1 4.97 5.69 5.00 5.64 23.66 4.22 5.92 6.99 3 4.32 4.83 4.13 4.00 4 4.00 4.01 3.36 2.91 5 4.274.22 9.66 8.48 6 2.83 3.20 8.60 9.25 7 4.46 4.79 5.80 6.40 8 4.04 4.784.02 4.63 9 2.98 3.10 10.02 10.16 10 4.88 5.04 6.24 6.44 11 2.95 3.297.28 8.43 12 4.47 4.97 6.75 7.70 13 2.75 3.01 4.91 4.62 14 4.35 4.738.48 9.27 15 3.82 4.16 6.26 6.06 16 3.16 3.50 14.49 14.97 17 3.87 4.225.37 4.67 18 3.69 4.04 3.75 3.50 19 4.08 4.51 11.42 11.22 20 3.03 3.262.00 1.87 21 3.23 3.49 6.68 6.50 22 4.35 4.63 10.09 9.95 23 2.84 3.0310.28 11.62 24 3.02 3.27 0.59 0.57 25 2.75 2.87 17.06 16.42 26 2.78 3.015.80 5.56 27 2.73 2.90 8.84 8.28 28 2.97 2.98 17.18 17.41 29 3.56 3.7516.70 16.79 30 2.91 3.16 7.05 7.89 31 3.32 3.55 9.80 9.73 32 3.01 3.2945.00 44.62 33 4.77 5.24 6.11 6.44 34 4.34 4.57 7.01 6.89 TABLE 1 showsthe raw data from counts performed on 34 vials. The second and thirdcolumns shows the red blood cell counts expressed as millions permicroliter of patient blood for the invention count and the Sysmexcount, respectively. The fourth and fifth columns shows the white bloodcell counts expressed as thousands per microliter of patient blood forthe invention count and the Sysmex count, respectively.

TABLE 2 Vial SysmexRBCs RBCcounts RBCscaled SysmexWBCs WBCcountsWBCscaled 1 5.69 1241765 4.97 5.64 1250 5.00 2 4.22 915262 3.66 6.991481 5.92 3 4.83 1080856 4.32 4.00 1033 4.13 4 4.01 998828 4.00 2.91 8403.36 5 4.22 1068411 4.27 8.48 2414 9.66 6 3.20 707250 2.83 9.25 21498.60 7 4.79 1115913 4.46 6.40 1451 5.80 8 4.78 1010933 4.04 4.63 10064.02 9 3.10 744241 2.98 10.16 2504 10.02 10 5.04 1220400 4.88 6.44 15596.24 11 3.29 736701 2.95 8.43 1819 7.28 12 4.97 1117506 4.47 7.70 16886.75 13 3.01 687645 2.75 4.62 1228 4.91 14 4.73 1086737 4.35 9.27 21208.48 15 4.16 955279 3.82 6.06 1564 6.26 16 3.50 789218 3.16 14.97 362214.49 17 4.22 967780 3.87 4.67 1343 5.37 18 4.04 922880 3.69 3.50 9373.75 19 4.51 1019878 4.08 11.22 2855 11.42 20 3.26 757606 3.03 1.87 5002.00 21 3.49 808679 3.23 6.50 1670 6.68 22 4.63 1086451 4.35 9.95 252210.09 23 3.03 709164 2.84 11.62 2571 10.28 24 3.27 753952 3.02 0.57 1470.59 25 2.87 688731 2.75 16.42 4265 17.06 26 3.01 695059 2.78 5.56 14515.80 27 2.90 682449 2.73 8.28 2209 8.84 28 2.98 741274 2.97 17.41 429517.18 29 3.75 890278 3.56 16.79 4174 16.70 30 3.16 727660 2.91 7.89 17627.05 31 3.55 831027 3.32 9.73 2450 9.80 32 3.29 753365 3.01 44.62 1125045.00 33 5.24 1193348 4.77 6.44 1527 6.11 34 4.57 1085941 4.34 6.89 17537.01 RBC Correlation (R{circumflex over ( )}2) 97.95% WBC Correlation(R{circumflex over ( )}2) 99.70% TABLE 2 shows the raw data from countsperformed on 34 vials. the 2^(nd) column gives the reference (Sysmex)RBC counts, while the 3^(rd) column reports the automated counts fromthe microscope slide. The 4^(th) column scales the counts to millioncells per microliter, assuming a 1:4 dilution. The 5^(th)-7^(th) columnshow the data for the WBC counts. At the bottom of the table are thecalculated correlation coefficients (R-squared).

The data was obtained from 34 patient samples during two sessions ofpreparing slides. The data is representative of typical patients,although the tubes were selected from patients with a wide distributionof red and white cell counts. Most, if not all of the 34 samples, wereobtained from specimens “archived” during the day in the refrigerator,and then pulled and prepared on the instrument in the late afternoon.Once the tubes were pulled, they were processed consecutively.

The algorithms were first validated by comparing manually countedmicroscope fields to the automated counts. There is a high correlationbetween the manually counted cells and the automatically counted cells.

High correlation between the two methods was found for both the redblood cell counts and the white blood cells counts (see Tables 1 and 2and FIGS. 5 and 6). The graph of FIG. 5 shows the correlation betweenthe Sysmex counts and the automated slide based counts for the red bloodcells. The data points are tightly clustered and form a line thatindicates that the numbers on the vertical axis (the invention counts)are similar to the numbers on the horizontal axis (the Sysmex counts).Typically for such data a correlation coefficient (R-squared) can becalculated to show the degree of agreement, where 100% would be perfectagreement. An R-squared value of 97.95% was calculated for this redblood cell data, indicating a high degree of agreement and similar towhat two different automated instruments might show. The graph shown inFIG. 6 shows the correlation between the Sysmex counts and the automatedslide counts for the white blood cells. The raw counts varied between147 and 11,250 white blood cells per slide. An R-squared value of 99.70%was calculated for this white blood cell data, indicating a high degreeof agreement and similar to what two different automated instrumentsmight show. This confirms that the novel approach to quantitativetransfer of cells was successful and that automated cell counts fromcomputer imaging yielded accurate results.

Exemplary Process for CBC and White Blood Cell (WBC) Differential

The following sequence of steps may be performed in any order and somesteps may be omitted or replaced with other steps.

-   Step 1. Extract a known volume of blood from a tube filled with a    patient's blood.-   Step 2. Dilute the blood if necessary. For example, one may use 5%    albumin in distilled water as a diluent.-   Step 3. Spread a known volume of blood or blood plus diluent over an    area on a glass microscope slide in a thin layer. The slide may be    treated to produce a hydrophilic surface to spread the cells better.    The slide may be treated to allow optimal adherence of the blood    elements to the slide.-   Step 4. Allow the slide to dry in the air, or assist the drying    using light air or heat.-   Step 5. Capture an image without a coverslip using a “dry” objective    that is corrected for no coverslip, for example one may use a 10× or    20× objective coupled to a CCD camera. Determine the count in each    image frame including Red Blood Cells (RBCs), and possibly White    Blood Cells (WBCs), and platelets. One or more colors may be used,    for example using a color camera or using narrow band illumination    produced by an interference filter or LED. Measurement of hemoglobin    content may be done at this time as well.-   Step 6. Fix and stain the cells on the slide. Fixation may be a    separate step or combined with staining.-   Step 7. Capture an image of stained slide without coverslipping,    using a “dry” objective, to count RBCs, WBCs, and platelets and    hemoglobin. This step may be in place of or in conjunction with step    5.-   Step 8. Perform WBC differential count from high resolution images    acquired without a coverslip, using a “dry” objective, for example    with a 40× or 50× objective that is not corrected for a coverslip. A    color camera or multiple black & white images taken using color    filters or using LED illumination may be used. This step may be in    addition to, or combined with Step 7.-   Step 9. Calculate desired parameters and derived parameters required    for the CBC.-   Step 10. Display all CBC parameters to an operator in a Graphical    User Interface (GUI).-   Step 11. Display results of WBC differential to an operator in the    GUI.-   Step 12. Display images of RBCs, WBCs, platelets and any    unusual/abnormal blood elements to an operator.-   Step 13. Allow an operator to interact with the images and the    parameters to “sign off” the CBC, WBC differential count, and    identification of unusual or abnormal objects.-   Step 14. If needed, update results of CBC and WBC counts depending    on operator interaction in Step 13.-   Step 15. Optionally, allow objects of interest to be relocated on a    microscope that has a motorized, computer controllable stage to    allow automated relocation of the objects for viewing.-   Step 16. Optionally, update the results of the CBC and WBC counts    depending on the microscopic operator interaction.

Although the exemplary process described above describes steps forpreparing and examining a sample of blood, embodiments of the inventionmay be used to prepare and examine other fluids comprising bone marrow,urine, vaginal tissue, epithelial tissue, tumors, semen, spittle, andother body fluids.

It is claimed:
 1. A system for applying cells from a blood sample onto asubstrate, the system comprising: a. an applicator tip for dispensingthe blood sample onto a substrate; b. a computer comprising amicroprocessor; c. software instructions stored on a storage device forcontrolling the system, wherein when the microprocessor executes thesoftware instructions, the computer causes the system to: i. fill theapplicator tip with a volume of the blood sample; ii. position theapplicator tip above the substrate; and iii. dispense a known volume ofblood sample out of the applicator tip, and while the ejection of theblood onto the substrate is occurring, maintain relative movementbetween the tip and the substrate to lay down the entire known volume ofblood sample in two or more rows over a defined area of the substratesuch that cells in the blood sample settle onto the substrate in a layerthat is about one cell thick, wherein morphology of the cells ispreserved sufficiently to enable image-based cell analysis; and d. alight receiving device for capturing an image of the cells on thesubstrate and wherein the computer causes the system to: i. capture animage of one of the rows of the blood sample; ii. analyze the image todetermine a width of the row of the blood sample; and ii. change aposition of the applicator tip relative to the substrate in a planeparallel to a surface of the substrate by an amount equal to the widthof the row of the blood sample.
 2. The system of claim 1, wherein thecomputer causes the system to image the blood cells on the substrate anddetermine one or more of a red blood cell count, white blood cell count,and a platelet count for the blood sample.
 3. The system of claim 1,wherein the computer causes the system to image the blood cells on thesubstrate and determine one or both of a complete blood count (CBC) anda white blood cell (WBC) differential for the blood sample.
 4. Thesystem of claim 1, wherein the computer controls movement of the tiprelative to the substrate by instructing the system to change theposition of the tip.
 5. The system of claim 1, wherein the systemfurther comprises a substrate controller and wherein the computercontrols movement of the tip relative to the substrate by instructingthe substrate controller to change the position of the substrate.
 6. Thesystem of claim 1, wherein the two or more rows comprise four or morerows, each row comprising a layer of cells about one cell thick, andwherein each of the second and subsequently dispensed rows settlesadjacent to a previously dispensed row.
 7. The system of claim 1,further comprising: a discharge unit for cleaning substrates; adispenser for dispensing stain onto substrates; a substrate tilter fortipping the substrate to remove excess stain; a substrate mover forpositioning the substrate under the light receiving device; and a lightsource for directing light onto or through the substrate.
 8. The systemof claim 1, wherein the substrate comprises a slide and the systemfurther comprises: a. a feeder for storing new slides; b. a firststation where the applicator applies the rows of the blood sample ontothe slide; c. a second station for staining, fixing, and drying theslide, d. a third station for illuminating and imaging the slide; e. acollector for receiving the slide; and f. an advancer for moving theslide through the system in order from the feeder to the first station,to the second station, to the third station, and to the collector. 9.The system of claim 1, further comprising a light source, wherein thecomputer causes the system to: a. generate light with the light sourceand use a first filter to filter the generated light to produce firstillumination light corresponding to a first wavelength range; b.illuminate the substrate with the first illumination light, and captureone or more images of the cells on the substrate; c. generate additionallight with the light source and use a second filter to filter theadditional light to produce second illumination light corresponding to asecond wavelength range; d. illuminate the substrate with the secondillumination light; and e. capture one or more images of the cells onthe substrate.
 10. The system of claim 9, wherein the computer causesthe system to: a. generate additional light with the light source, anduse a third filter to filter the additional light to produce thirdillumination light corresponding to a third wavelength range; b.illuminate the substrate with the third illumination light; and c.capture one or more images of the cells on the substrate.
 11. The systemof claim 9, wherein the first wavelength range is between about 400 nmand 470 nm, and the second wavelength range is between about 470 nm and750 nm.
 12. The system of claim 10, wherein specific wavelengths fromthe first, second, and third wavelength ranges are selected to be 430nm, 500 nm, 525 nm, and 600 nm.
 13. The system of claim 9, wherein thecomputer causes the system to: a. refine the images by sharpening orcompensating for spatial shifts or other distortions; and b. combine twoor more images of the cells to generate a multi-color image, wherein thecombined images comprise at least one image corresponding to the firstillumination light at the first wavelength range, and at least one imagecorresponding to the second illumination light at the second wavelengthrange.
 14. The system of claim 9, wherein the computer causes the systemto determine one or more of spatial, densitometric, colorimetric, andtexture features of the cells from the images for classification of acell type.
 15. The system of claim 1, wherein the system furthercomprises a light source comprising multiple light emitting diodes(LEDs), and wherein the computer causes the system to: a. activate afirst LED in the light source to generate first illumination light at afirst wavelength range; b. illuminate the substrate with the firstillumination light, and use the light receiving device to capture one ormore images of the cells on the substrate; c. activate a second LED inthe light source to generate second illumination light at a secondwavelength range; and d. illuminate the substrate with the secondillumination light, and use the light receiving device to capture one ormore images of the cells on the substrate, wherein before or duringillumination of the substrate with the second illumination light, thecomputer causes the system to adjust a focal position of lighttransmitted or reflected by the rows of the blood sample so that thetransmitted or reflected light is focused onto the light receivingdevice.
 16. The system of claim 1, wherein the two or more rows arenon-touching rows.
 17. The system of claim 1, wherein the two or morerows are formed by the applicator tip dispensing a first row of theblood while moving relative to the substrate along a first direction ina plane parallel to the substrate, and then turning and moving in asecond direction opposite and parallel to the first direction to laydown a second row adjacent to the first row.
 18. The system of claim 1,wherein the software causes the system to: a. dispense the blood fromthe applicator tip at a rate of about 0.1 microliters per second; and b.maintain a speed of relative movement between the tip and the substrateof about 10 to 100 millimeters per second.
 19. The system of claim 1,wherein the two or more rows are laid down on the substrate and settleadjacent one another to form a contiguous layer of cells in the definedarea.
 20. The system of claim 1, wherein the system further comprises alight source arranged to separately emit a first light at a wavelengthrange of 400 nm to 470 nm and a second light at a wavelength range of470 nm to 750 nm onto the rows of the blood sample on the substrate. 21.The system of claim 20, wherein the first light has a wavelength ofabout 430 nm and the second light has a wavelength of about 600 nm. 22.The system of claim 15, wherein the first wavelength range is betweenabout 400 nm and 470 nm, and the second wavelength range is betweenabout 470 nm and 750 nm.
 23. The system of claim 15, wherein thecomputer further causes the system to: a. activate a third LED in thelight source to generate third illumination light at a third wavelengthrange; and d. illuminate the substrate with the third illuminationlight, and use the light receiving device to capture one or more imagesof the cells on the substrate.
 24. The system of claim 23, wherein thefirst, second, and third wavelengths are selected from the group ofwavelengths consisting of 430 nm, 500 nm, 525 nm, and 600 nm.
 25. Thesystem of claim 2, further comprising: a display; and a dispenserconfigured to apply a stain solution and a fixative solution to thesubstrate, wherein the computer causes the system to: show the images ofthe cells captured by the light receiving device on the display; analyzethe images of the cells to determine a reticulocyte count for the bloodsample; and show the reticulocyte count and the one or more of a redblood cell count, white blood cell count, and a platelet count for theblood sample on the display.
 26. The system of claim 2, furthercomprising: a display; a dispenser configured to apply a stain solutionand a fixative solution to the substrate; a gas movement deviceconfigured to expose the substrate to a stream of flowing gas; and asubstrate advancer configured to transport the substrate in order fromthe applicator to the dispenser and then to the light receiving device,wherein the computer causes the system to: show the images of the cellscaptured by the light receiving device on the display; use the gasmovement device to control a rate of gas flow to which the substrate isexposed to adjust a drying time of the cells on the substrate; analyzethe images of the cells to determine a reticulocyte count for the bloodsample; and show the reticulocyte count and the one or more of a redblood cell count, white blood cell count, and a platelet count for theblood sample on the display.